Alterations in nuclear structure are associated with aging and cancer, and yet the functional link between nuclear architecture and nuclear functions is not well understood. We have been using budding yeast as a model system to study nuclear architecture. The yeast nucleus differs from that of higher eukaryotes in two aspects: (1) yeast lack lamins, proteins that play a major structural role in shaping the nucleus in cells of metazoans. There is also accumulating evidence to suggest that lamins contribute to various nuclear processes. (2) The yeast nuclear membrane remains intact throughout the cell cycle, unlike nuclear membrane of higher eukaryotes, which breaks down during mitosis. Nonetheless, in an earlier study from our lab (Campbell et al, 2006), we showed that the shape of the yeast nucleus is determined by three factors: the composition of the nuclear membrane, the shape of the chromatin, and the presence of an unidentified nuclear structure that tethers the nuclear membrane to the chromatin, akin to nuclear lamins of higher eukaryotes. Our previous study focused on a yeast strain in which the Spo7 protein was inactivated. Spo7 is a regulator of phospholipids synthesis; in its absence phospholipids levels increase, leading to the expansion of the endoplasmic reticulum (ER) and certain regions of the nucleus. In particular, we were able to show that only the membrane associated with the nucleolus (a sub-compartment of the nucleus) expands, whereas the rest of the nuclear membrane remains juxtaposed to the bulk of the chromatin. This led to the hypothesis that in yeast there is a nuclear tether that associates the nuclear membrane to the chromatin and resists membrane expansion when phospholipids levels increase. Based on this hypothesis we assume that inactivating this tether will further alter nuclear shape, to the point where nuclear functions will be severely compromised, and this will be reflected in reduced cell viability. Thus, we sought mutations whose combination with a spo7 mutation leads to cell death or severe growth defects. Two approaches were used to find such mutations: in the first we conducted a screen for randomly induced mutations that cause cell death in a yeast background lacking Spo7 function (known as a yeast synthetic lethal screen). This was followed by a secondary screen for mutants that have an altered nuclear shape. In a pilot screen we have identified two mutations that lead to an altered nuclear shape, one of which is in a nucleolar protein. We are currently expanding the screen to uncover more proteins that contribute to nuclear shape. Once identified, we will study how these proteins affect nuclear shape and what role they play in coordinating nuclear architecture with nuclear function.[unreadable] [unreadable] In the second approach we combined mutations in candidate genes with the spo7 mutation, looking for reduced growth in the double mutant. The candidate genes were selected based on their function and localization; most were involved in processes that take place near or at the nuclear membrane and most exhibited a peripheral localization. Through this screen we found one mutant that showed a severe growth defect when combined with a spo7 mutation. This mutant was known to affect spindle pole body function, and therefore we tested additional mutations known to abrogate spindle pole body assembly. We found that in many cases the spindle pole body defect was exacerbated when Spo7 was also absent. These findings suggest that the composition of the nuclear membrane affects the function of associated structures. We are now collaborating with Dr. Sue Jaspersen in order to look at spindle pole bodies by electron microscopy and to determine the relationship between nuclear membrane composition and the assembly of integral membrane structures.